Scalable purification method for aav9

ABSTRACT

A two-step chromatography purification scheme is described which selectively captures and isolates the genome-containing rAAV vector particles from the clarified, concentrated supernatant of a rAAV production cell culture. The process utilizes an affinity capture method performed at a high salt concentration followed by an anion exchange resin method performed at high pH to provide rAAV vector particles which are substantially free of rAAV intermediates.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under W911NF-13-2-0036awarded by Defense Advanced Research Projects Agency (DARPA). Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

This invention describes a novel scalable method for producing rAAVsuitable for clinical applications. Also provided are purified rAAV. Theuse of recombinant adeno-associated viruses (rAAV) for a variety of genetherapy and vaccine approaches have been described. However, even withthese approaches, scalable methods for purification of rAAV have beenlacking.

Adeno-associated virus (AAV), a member of the Parvovirus family, is asmall, non-enveloped virus. AAV particles comprise an AAV capsidcomposed of 60 capsid protein subunits, VP1, VP2 and VP3, which enclosea single-stranded DNA genome of about 4.7 kilobases (kb). These VP1, VP2and VP3 proteins are present in a predicted ratio of about 1:1:10, andare arranged in an icosahedral symmetry. Individual particles packageonly one DNA molecule strand, but this may be either the plus or minusstrand. Particles containing either strand are infectious. AAV isassigned to the genus, Dependovirus, because the virus was discovered asa contaminant in purified adenovirus stocks. AAV's life cycle includes alatent phase and an infectious phase. Replication occurs by conversionof the linear single stranded DNA genome to a duplex form, andsubsequent amplification, from which progeny single strands are rescued,replicated, and packaged into capsids in the presence of helperfunctions. The properties of non-pathogenicity, broad host range ofinfectivity, including non-dividing cells, and integration make AAV anattractive delivery vehicle.

Recombinant AAV particles are produced in permissive (packaging) hostcell cultures and co-expression of helper virus AAV rep and AAV capgenes are required, for replication and packaging, the recombinantgenome into the viral particle. Genes necessary for genome replication,capsid formation and genome packaging can be expressed from transfectedplasmids, integrated into the host cell genome or introduced to the cellby recombinant viruses. Typically, cells are lysed to release rAAVparticles and maximize yield of recovered rAAV. However, the cell lysatecontains various cellular components such as host cell DNA, host cellproteins, media components, and in some instances, helper virus orhelper virus plasmid DNA, which must be separated from the rAAV vectorbefore it is suitable for in vivo use. Recent advances in rAAVproduction include the use of non-adherent cell suspension processes instirred tank bioreactors and production conditions whereby rAAV vectorsare released into the media or supernatant reducing the concentration ofhost cellular components present in the production material but stillcontaining appreciable amounts of in-process impurities. See U.S. Pat.No. 6,566,118 and PCT WO 99/11764. Therefore, rAAV particles may becollected from the media and/or cell lysate and further purified.

Certain previously described purification methods for rAAV are notscalable and/or not adaptable to good manufacturing practices,including, e.g., cesium chloride gradient centrifugation and iodixanolgradient separation. See, e.g., M. Potter et al, MolecularTherapy—Methods & Clinical Development (2014), 1: 14034, pp 1-8.

US Patent Publication No. 2005/0024467 reports that rAAV capsidserotypes such as rAAV-1, 4, 5, and 8 bind weakly to anionic resinseither as purified virus stock or in the presence of in-processproduction impurities such as host cell DNA, host cell proteins, serumalbumin, media components, and helper virus components.

Purification of those capsid serotypes is described as involvinganion-exchange chromatography in combination with other purificationmethods, such as iodixinol density-gradient centrifugation. See, e.g.,Zolotukhin et al., Methods 28(2):158-167 (2002) and Kaludov et al., Hum.Gene Therapy 13:1235-1243 (2002); and U.S. Patent Publication No.2004/0110266 A1. However, those methods are not readily scalable tocommercial scale processes.

Other examples of one- or two-step ion-exchange chromatographypurification have been reported for rAAV serotypes 1, 2, 4, 5, and 8.[Brument, N, et al. (2002). Mol Ther 6: 678-686; Okada, T, et al.(2009). Hum Gene Ther 20: 1013-1021; Kaludov, N, et al (2002). Hum GeneTher 13: 1235-1243; Zolotukhin, S, et al. (2002). Methods 28: 158-167;Davidoff, A M, et al. (2004). J Virol Methods 121: 209-215]. Morerecently, an affinity media incorporating an anti-AAV VHH ligand, asingle-domain camelid antibody derivative, was described as being usefulto purify serotypes 1, 2, 3, and 5. [Hellström, M, et al. (2009) GeneTher 16: 521-532]. This affinity capture method focuses on purifyingrAAV vectors from in-process production components of the cell cultureincluding helper virus, as well as helper virus proteins, cellularproteins, host cell DNA, and media components present in the rAAVproduction stock. The affinity capture method described for purifyingrAAV 1, 2, 3 and 5 particles is designed to purify rAAV from host celland helper virus contaminants, but not to separate AAV particles fromempty AAV capsids lacking packaged genomic sequences. Further, it is notclear from the literature that this separation is desirable. See, e.g.,F. Mingozzi et al, Sci Transl med. 2013 Jul. 17: 5(194), avail in PMC2014 Jul. 14, which suggests it may be desirable to include emptycapsids as decoys which can be used to overcome preexisting humoralimmunity to AAV can be overcome using capsid decoys. However, otherauthors have reported increase efficacy in rAAV1 vectors when they wereseparated from empty AAV1 capsids. See, e.g., M. Urabe et al, MolecularTherapy, 13(4):823-828 (April 2006).

There remains a need for scalable methods for separatingpharmacologically active (full) rAAV particles having the desiredtransgene packaged from rAAV capsids which lack the desired transgene.

SUMMARY OF THE INVENTION

The present invention provides a scalable method for efficientlyseparating genome-containing AAV9 vector particles (full) fromgenome-deficient rAAV9 intermediates (empty capsids).

In one aspect, the method for separating full AAV9 viral particles fromempty AAV9 intermediates comprises subjecting a mixture comprisingrecombinant AAV9 viral particles and AAV9 vectorintermediates/byproducts to fast performance liquid chromatography(FPLC), wherein the AAV9 viral particles and AAV9 intermediates arebound to a strong anion exchange resin equilibrated at a pH of about10.2 and subjected to a salt gradient while monitoring the eluate forultraviolet absorbance at about 260 nm and about 280 nm. The AAV9 fullcapsids are collected from a fraction which is eluted when the ratio ofA260/A280 reaches an inflection point. More particularly, the fullcapsids are collected from the eluted fraction(s) characterized byhaving a higher peak (area under the curve) at an absorbance of 260nm ascompared to the peak (area under the curve) at an absorbance of 280 nm.The majority of the fractions observed for the process described hereinhave a higher amount of empty capsids (higher peak/area under curve atA280). The absorbance peak at 260 nm being equal to or exceeding theabsorbance peak at 280 nm is indicative of the fraction containing thefull capsids.

In a further aspect, the sample loaded into the fast protein liquidchromatography (FPLC) method contains full recombinant AAV9 viralparticles and AAV9 intermediates (empty capsids) that had been purifiedfrom production system contaminants using affinity capture. In oneembodiment, the affinity capture is performed using a high performanceaffinity resin having an antibody specific for AAV.

In still another aspect, a scalable method is provided for separatingfull AAV9 viral particles from AAV9 intermediates by using an anti-AAVantibody based affinity capture resin followed by an anion exchangeresin. In one embodiment, the mixture containing the AAV9 viralparticles and AAV9 intermediates is loaded onto the affinity resin in abuffer having a high salt concentrations, e.g., about 400 nM NaCl toabout 650 mM NaCl or another salt(s) having an equivalent ionicstrength. The wash step for the affinity resin is thereafter performedat an even higher salt concentration, e.g., in the range of about 750 mMto about 1 M NacCl or equivalent. In one embodiment, the AAV9 mixture ismaintained at a salt concentration of about 400 mM NaCl to about 650 mMNaCl, or equivalent prior to being applied to the anion exchange resincolumn.

In yet another aspect, a method for separating AAV9 viral particles fromAAV9 capsid intermediates is provided, said method comprising: (a)mixing a suspension comprising recombinant AAV9 viral particles and AAV9 vector intermediates and a Buffer A comprising 20 mM to 50 mM Bis-Trispropane (BTP) and a pH of about 10.2 (e.g., 10.2); (b) loading thesuspension of (a) onto a strong anion exchange resin column; (c) washingthe loaded anion exchange resin with Buffer 1% B which comprises a salthaving the ionic strength of 10mM to 40 mM NaCl and BTP with a pH ofabout 10.2 (e.g., 10.2); (d) applying an increasing salt concentrationgradient to the loaded and washed anion exchange resin, wherein the saltgradient is the equivalent of 10 mM to about 40 mM NaCl; and (e)collecting rAAV9 particles from the eluate at the inflection point, saidrAAV particles being purified away from AAV9 intermediates. In oneembodiment, the rAAV9 particles are at least about 90% purified fromAAV9 intermediates. In one embodiment, the fractions with the highestconcentration of “full” rAAV9 particles elute at a salt concentrationlower than the fractions with empties and other intermediates.

In a further aspect, a scalable method is provided for separatingpharmacologically active recombinant AAV9 viral particles containing DNAgenomic sequences from inert genome-deficient (empty) AAV9 vectorintermediates, said method comprising: (a) forming a loading suspensioncomprising: recombinant AAV9 viral particles and empty AAV9 capsid whichhave been purified to remove contaminants from an AAV producer cellculture in which the particles and intermediates were generated; and aBuffer A comprising 20 mM Bis-Tris propane (BTP) and a pH of about 10.2;(b) loading the suspension of (a) onto a strong anion exchange resin,said resin being in a vessel having an inlet for flow of a suspensionand/or solution and an outlet permitting flow of eluate from the vessel;(c) washing the loaded anion exchange resin with Buffer 1% B whichcomprises 10 mM NaCl and 20 mM BTP with a pH of about 10.2; (d) applyingan increasing salt concentration gradient to the loaded and washed anionexchange resin, wherein the salt gradient ranges from 10 mM to about 190mM NaCl, inclusive of the endpoints, or an equivalent ; and (e)collecting the rAAV particles from eluate collected at the inflectionpoint, said rAAV particles being purified away from AAV9 intermediates.

In a further aspect, the affinity resin separation comprises: (i)equilibrating the affinity resin with Buffer A1 which comprises about200 mM to about 600 mM NaCl, about 20 mM Tris-Cl and a neutral pH priorto applying the material to the affinity resin; (ii) washing the loadedresin of (a) with Buffer C1 which comprises about 800 mM NaCl to about1200 mM NaCl, 20 mM Tris-Cl and a neutral pH; (iii) washing the BufferC1-washed resin of (b) with Buffer A1 to reduce salt concentration; (iv)washing the affinity resin of (c) with Buffer B which comprises about200 nM to about 600 nM NaCl, 20 mM Sodium Citrate, pH about 2.4 to about3; and (v) collecting the eluate of (iv) which comprises the full AAV9particles and the empty AAV9 capsid fraction for loading onto the anionexchange resin.

In still another aspect, vector preparations are provided that have lessthan 5% contamination with AAV intermediates (including AAV emptycapsids). In another aspect, vector preparations are provided that haveless than 2% contamination with AAV empty capsids, or less than 1%contamination with AAV empty capsids. In a further aspect, AAVcompositions are provided which are substantially free of AAV emptycapsids.

Still other advantages of the present invention will be apparent fromthe detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D illustrate the results of a deconstructed method developmentfor separation of AAV9 vector particle populations on AEX monolithcolumns. FIG. 1A is a chromatogram of a CIMac QA™ (commerciallyavailable from Biaseparations) column run. 3×10¹² GC full vectorparticles purified on an iodixanol gradient were loaded to a 0.1 mLCIMac QA™ column at pH10.2 and eluted with a 60 column volume (CV) 10mM-180mM linear salt gradient at a flow rate of 2 mL/min. A260 (lineextending highest at peak P2), A280 (line extending second highest atpeak P2), programmed salt gradient (polyline extending from y axis andreaching ˜31 mAU after the run volume exceeds 25 mL) and conductivity(smooth line reaching ˜6 mAU when the run volume is ˜25 mL) profiles areshown. The y-axis absorbance units are in mAU. Run volume (mL) is shownas solid line beneath the x axis while buffer is indicated on the x axisabove the run volume. The major peak observed in the salt gradient islabeled P2. FIG. 1B provides a nominal volume (100 μL) of CsClgradient-purified empty AAV9 particles was loaded to a 0.1 mL CIMac QA™column at pH10.2 and eluted with a linear salt gradient. Profiles andaxis units are identical to those described for FIG. 1A. The major peaksobserved in the salt gradient and high salt column wash fractions arelabeled P1, P3 and P4. In FIG. 1B, the A280 peak is higher than the A260peak in each of P1, P3 and P4. Programmed salt gradient is shown as apolyline extending from y axis and reaching ˜5.7 mAU after the runvolume exceeds 25 mL and conductivity is shown as a smooth line reaching˜1 mAU when the run volume is ˜25 mL. FIG. 1C is an overlay of the A280traces from FIGS. 1A (line extending highest at peak P2) and 1B (lineextending highest at peaks P3-P4) and the major peaks observed in thesalt gradient and high salt column wash fractions are labeled P1- P4.FIG. 1D shows the same empty particle (100 uL) and full particle (3×10¹²GC) preparations run in FIGS. 1A and 1B were mixed prior to loading to a0.1 mL CIMac QA column at pH10.2 and eluting with a linear saltgradient. The major peaks observed in the salt gradient and high saltcolumn wash fractions are labeled P1-P4. A260 (line extending highest atpeak P2 and second highest at peaks P3-P4), A280 (line extending secondhighest at peak P2 and highest at peaks P3-P4), programmed salt gradient(polyline extending from y axis and connecting with y axis at ˜1 mAU)and conductivity (smooth line extending from y axis and connecting withy axis at ˜0 mAU) profiles are shown. The y-axis absorbance units are inmAU. Run volume (mL) is shown as solid line beneath the x axis whilebuffer is indicated on the x axis above the run volume.

FIG. 2 shows the purification of an affinity purified AAV9 vectorpreparation by anion exchange chromatography on a monolith anionexchange resin (AEX) column. A PorosAAV9™ [ThermoFischerScientific]affinity-purified AAV9 vector preparation (6×10¹² GC) was run on a 0.1mL CIMac QA™ column and the chromatogram is shown. The run was performedwith 20 mM Bis-Tris-Propane (BTP) pH 10.2 as the loading buffer (bufferA) and 20 mM BTP pH 10.2-1M NaCl as the column strip buffer (Buffer B).A 60 CV linear salt gradient from 1-18% Buffer B was used to elutevector and the column was stripped with 100% Buffer B. The flow rate wasmaintained at 2 mL/min throughout the run. A260 (line extending highestat peak P2 and second highest at peaks P3 and P4), A280 (line extendingsecond highest at peak P2 and highest at peaks P3 and P4), programmedconductivity (polyline extending from y axis and reaching ˜106 mAU afterthe run volume exceeds 55 mL) and actual conductivity (smooth lineextending from y axis and reaching ˜20 mAU when the run volume is around55 mL) profiles are shown. Absorbance (mAU) is shown on the y axis. Runvolume (mL) is shown as solid line beneath the x axis while buffer isindicated on the x axis above the run volume. The major peaks (labelledP1-P4) are indicated. GC recoveries for the major peaks are given belowthe chromatogram.

FIGS. 3A-3B shows the reproducibility of chromatogram peak distributionand A260/280 ratios for an affinity purified AAV9 vector preparation runat increased scale on a monolith AEX column. FIG. 3A shows achromatogram from a PorosAAV9™ affinity-purified AAV9 vector preparation(5×10¹⁴ GC) run on an 8 mL CIMmultus QA™ column. The run was performedwith 20 mM Bis-Tris-Propane (BTP) pH10.2 as the loading buffer (bufferA) and 20 mM BTP pH10.2-1M NaCl as the column strip buffer (Buffer B). A60 CV linear salt gradient from 1-19% Buffer B was used to elute vectorand the column was stripped with 100% Buffer B. The flow rate wasmaintained at 20 mL/min throughout the run. A260 (line extending highestat peak P2 and second highest at peaks P3 and P4), A280 (line extendingsecond highest at peak P2 and highest at peaks P3 and P4), programmedconductivity (polyline extending from y axis and reaching ˜243 mAU afterthe run volume exceeds ˜1345 mL) and actual conductivity (smooth lineextending from y axis and reaching ˜45 mAU when the run volume is ˜1345mL) profiles are shown. Absorbance (mAU) is shown on the y axis and runvolume (mL) on the x axis. The major peaks (labelled P1-P4) areindicated. FIG. 3B shows SDS PAGE-based particle quantification of peaksP1-P4. SDS PAGE gels were loaded with serial dilutions of an iodixanolgradient-purified “full” reference standard alongside similar dilutionsof peak fractions. The capsid protein VP3 band was quantified for eachdilution and a standard curve of particle number loaded versus bandvolume obtained. Particle (pt) numbers for each peak fraction weredetermined by comparison of band volumes to the standard curve. pt: GCratios and percent empty capsids for the peaks were derived bycomparison of GC loaded and the determined pt number.

DETAILED DESCRIPTION OF THE INVENTION

This invention allows a scalable technology for production of purifiedrAAV9 for use in a variety of gene transfer and/or other applications.Suitably, the method purifies rAAV9 viral particles from productionculture contaminants such as helper virus, helper virus proteins,plasmids, cellular proteins and DNA, media components, serum proteins,AAV rep proteins, unassembled AAV VP1, AAV VP2 and AAV VP3 proteins, andthe like. Further, the method provided herein is particularly wellsuited for separating full rAAV9 viral particles from rAAVintermediates.

In one aspect, the method for separating full AAV9 viral particles fromempty AAV9 intermediates comprises subjecting a mixture comprisingrecombinant AAV9 viral particles and AAV 9 vector intermediates to fastperformance liquid chromatography, wherein the AAV9 viral particles andAAV9 intermediates are bound to a strong anion exchange resinequilibrated at a pH of about 10.2 (e.g., 10.0 to 10.4, preferably 10.2)and subjected to a salt gradient while monitoring eluate for ultravioletabsorbance at about 260 nm and about 280 nm, respectively.

More particularly, the presence of AAV9 capsids having genomic sequencespackaged therein (“full”) and 260/280 absorbance ratios less than 1 ischaracteristic of AAV9 intermediates as defined herein. In general, theproduction cell culture may yield a mixture of rAAV9 “full” and rAAV9“empty” or other intermediates in which 50% or greater are intermediates(including empties), at least 60% are intermediates, or greater than 70%are intermediates. In other embodiments, more or less of the genomecopies are “empty”; as a consequence, a corresponding amount of elutedfractions are characterized by having 280 nm peaks (and correspondinglarger areas under the curve which are larger than 260 nm peaks).Fractions characterized by peaks (and corresponding larger areas underthe curve) at an absorbence of about 260 nm (A260) that are higher thanthe corresponding peaks at 260 nm (A260/280 ratio is >1) are highlyenriched in full rAAV9 particles. The AAV9 full capsids are collectedfrom a fraction which is eluted when the peak for A260 crosses over andexceeds the peak for A280 (i.e., reaches an inflection point).

As used herein, “recombinant AAV9 viral particle” refers tonuclease-resistant particle (NRP) which has an AAV9 capsid, the capsidhaving packaged therein a heterologous nucleic acid molecule comprisingan expression cassette for a desired gene product. Such an expressioncassette typically contains an AAV 5′ and/or 3′ inverted terminal repeatsequence flanking a gene sequence, in which the gene sequence isoperably linked to expression control sequences. These and othersuitable elements of the expression cassette are described in moredetail below and may alternatively be referred to herein as thetransgene genomic sequences. This may also be referred to as a “full”AAV capsid. Such a rAAV viral particle is termed “pharmacologicallyactive” when it delivers the transgene to a host cell which is capableof expressing the desired gene product carried by the expressioncassette.

In many instances, rAAV particles are referred to as DNase resistant(DRP). However, in addition to this endonuclease (DNase), exonucleasesmay also be used in the purification steps described herein, to removecontaminating nucleic acids. Such nucleases may be selected to degradesingle stranded DNA and/or double-stranded DNA, and RNA. Such steps maycontain a single nuclease, or mixtures of nucleases directed todifferent targets, and may be endonucleases or exonucleases.

The term “nuclease-resistant” indicates that the AAV capsid has fullyassembled around the expression cassette which is designed to deliver atransgene to a host cell and protects these packaged genomic sequencesfrom degradation (digestion) during nuclease incubation steps designedto remove contaminating nucleic acids which may be present from theproduction process.

As used herein, “AAV9 capsid” refers to the AAV9 having the amino acidsequence of GenBank accession:AAS99264, which is incorporated byreference herein and reproduced in SEQ ID NO: 1. In addition, themethods provided herein can be used to purify other AAV having a capsidhighly related to the AAV1 capsid. For example, AAV having about 99%identity to the referenced amino acid sequence in GenBankaccession:AAS99264 and U.S. Pat. No. 7,906,111 (also WO 2005/033321)(i.e., less than about 1% variation from the referenced sequence) may bepurified using the methods described herein, provided that the integrityof the ligand-binding site for the affinity capture purification ismaintained and the change in sequences does not substantially alter thepH range for the capsid for the ion exchange resin purification. SuchAAV may include, e.g., natural isolates (e.g., hu31 or hu32), orvariants of AAV9 described, e.g., in U.S. Pat. No. 9,102,949, U.S. Pat.No. 8,927,514, US2015/349911; and WO 2016/049230A1. Methods ofgenerating the capsid, coding sequences therefore, and methods forproduction of rAAV viral vectors have been described. See, e.g., Gao, etal, Proc. Natl. Acad. Sci. U.S.A. 100 (10), 6081-6086 (2003) and US2013/0045186A1.

The term “identity” or “percent sequence identity” may be readilydetermined for amino acid sequences, over the full-length of a protein,a subunit, or a fragment thereof. Suitably, a fragment is at least about8 amino acids in length, and may be up to about 700 amino acids.Examples of suitable fragments are described herein. Generally, whenreferring to “identity”, “homology”, or “similarity” between twodifferent adeno-associated viruses, “identity”, “homology” or“similarity” is determined in reference to “aligned” sequences.“Aligned” sequences or “alignments” refer to multiple nucleic acidsequences or protein (amino acids) sequences, often containingcorrections for missing or additional bases or amino acids as comparedto a reference sequence. Alignments are performed using a variety ofpublicly or commercially available Multiple Sequence Alignment Programs.Examples of such programs include, “Clustal W”, “CAP Sequence Assembly”,“MAP”, and “MEME”, which are accessible through Web Servers on theinternet. Other sources for such programs are known to those of skill inthe art. Alternatively, Vector NTI utilities are also used. There arealso a number of algorithms known in the art that can be used to measurenucleotide sequence identity, including those contained in the programsdescribed above. As another example, polynucleotide sequences can becompared using Fasta™, a program in GCG Version 6.1. Fasta™ providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. For instance, percentsequence identity between nucleic acid sequences can be determined usingFasta™ with its default parameters (a word size of 6 and the NOPAMfactor for the scoring matrix) as provided in GCG Version 6.1, hereinincorporated by reference. Multiple sequence alignment programs are alsoavailable for amino acid sequences, e.g., the “Clustal X”, “MAP”,“PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs.Generally, any of these programs are used at default settings, althoughone of skill in the art can alter these settings as needed.Alternatively, one of skill in the art can utilize another algorithm orcomputer program which provides at least the level of identity oralignment as that provided by the referenced algorithms and programs.See, e.g., J. D. Thomson et al, Nucl. Acids Res., “A comprehensivecomparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

As used herein the term “AAV9 intermediate” or “AAV9 vectorintermediate” refers to an assembled rAAV capsid which lacks genomicsequences packaged therein. These may also be termed an “empty” capsid.Such a capsid may contain no detectable genomic sequences of anexpression cassette, or only partially packaged genomic sequences whichare insufficient to achieve expression of the gene product. These emptycapsids are non-functional to transfer the gene of interest to a hostcell.

In one aspect, a method for separating rAAV9 particles having packagedgenomic sequences from genome-deficient AAV9 intermediates is provided.This method involves subjecting a suspension comprising recombinant AAV9viral particles and AAV 9 capsid intermediates to fast performanceliquid chromatography, wherein the AAV9 viral particles and AAV9intermediates are bound to a strong anion exchange resin equilibrated ata pH of 10.2, and subjected to a salt gradient while monitoring eluatefor ultraviolet absorbance at about 260 and about 280. Although lessoptimal for rAAV9, the pH may be in the range of about 10.0 to 10.4. Inthis method, the AAV9 full capsids are collected from a fraction whichis eluted when the ratio of A260/A280 reaches an inflection point.

Fast protein liquid chromatography (FPLC), is a form of liquidchromatography that is often used to analyze or purify mixtures ofproteins. As in other forms of chromatography, separation is possiblebecause the different components of a mixture have different affinitiesfor two materials, a moving fluid (the “mobile phase”) and a poroussolid (the stationary phase). In the present invention, the mobile phaseis an aqueous solution, or “buffer”. The buffer flow rate may becontrolled by gravity or a pump (e.g., a positive-displacement pump) andcan be kept constant or varied. Suitably, the composition of the buffercan be varied by drawing fluids in different proportions from two ormore external reservoirs. The stationary phase described herein is astrong anion exchange resin, typically composed of beads. These beadsmay be packed into a vessel, e.g., a cylindrical glass or plasticcolumn, or another suitable vessel. As provided herein, volumes of themobile phase are described as “column volumes”. These volumes may beextrapolated to other vessel shapes and designs.

The eluate from the anion exchange resin column or other vessel ismonitored for ultraviolet absorbance at about 260 nm and 280 nm. Asprovided herein, “full” AAV9 capsids are characterized by having a UVabsorbance of about 260 nm, whereas as “empty” capsids are characterizedby having a UV absorbance of about 280 nm. Typically, the majority ofthe eluate fractions contain empty capsids and as the salt gradientprogresses, the majority of the eluate is characterized by a curve for

A280 exceeding that of A260. By monitoring UV absorbance for when theeluate is characterized by the curve for A260 crossing over the curvefor A280 (ratio of A260/A280 greater than 1), one can selectivelycollect the “full capsids” until such time as the ratio reverts toA280/A260 greater than 1.

In one embodiment, this fraction(s) selectively collected at theinflection point is characterized by having the total collected rAAVcontain at least about 90% “full capsids”, and preferably, at least 95%“full capsids”. In a further embodiment, these fractions may becharacterized by having a ratio of “intermediate” to “full” less than0.75, more preferably 0.5, preferably less than 0.3.

As used herein, an “anion exchange resin” refers to an insoluble matrixor solid support (e.g., beads) capable of having a surface ionizationover a pH range of about 1 to about 14. In one embodiment, a stronganion exchange resin is a solid support having a surface coated withquaternized polyethyleneimine. An example of such a strong anionicexchange resin is the solid support of the CIMultus QA™ column. Forexample, the anion exchange resin may be a quaternary amine ion exchangeresin. In a further embodiment, the anion exchange resin comprisestrimethylamine and a support matrix comprising poly(glycidylmethacrylate-co-ethylene dimethacrylate). However, other suitable anionexchange resins may be selected. An example of such a strong anionicexchange resin is that of the POROS HQ™ column. The resins for thecolumns listed above can be obtained from Amersham/Pharmacia(Piscataway, N.J.), PerSeptive Biosystems (Foster City, Calif.),TosoHaas (Montgomeryville, Pa.) and other suppliers.

The anion exchange material may be in the form of a monolith column or atraditional bead-based column. The ion exchange material can be in acolumn having a capacity of 0 to 0.5 mL column, 1 mL column, and morepreferably, at least an 8 mL column, a 10 mL column, a 20 mL column, a30 mL column, a 50 mL column, a 100 mL column, a 200 mL column, a 300 mLcolumn, a 400 mL column, a 500 mL column, a 600 mL column, a 700 mLcolumn, an 800 mL column, a 900 mL column, a 1000 mL (1L) column, a 2000mL (2L) column, a 10 L column, a 20 L column, a 30 L column, a 40 Lcolumn, a 50 L column, a 60 L column, a 70 L column, an 80 L column, a90 L column, a 100L column, a 140 L column, or a column with a capacitygreater than 140 L as well as any other column with a capacity betweenthe volumes listed above. Alternatively, another vessel type may be usedto contain the anion exchange resin solid support.

As shown in the examples, regulation of the loading and flow rateenhances separation of the empty and full capsids. In one embodiment,the sample loading flow rate is less than or equal to the elution flowrate. For example, the loading flow rate may be in the range of about 10mL/min to about 40 mL/min, about 15 mL/min to about 30 mL/min, or about20 mL/min to about 25 mL/min, about 10 mL/min, about 20 mL/min, or about30 cm/hr to about 135 cm/hr, for a 8 mL monolith column. Suitable flowrates may be extrapolated for a non-monolith column.

The specification describes salt concentrations herein with reference toNaCl for convenience. However, it will be understood that another saltof an equivalent ionic strength (e.g., KCl) may be substituted therefor,another salt having a different ionic strength, but its concentrationadjusted to an equivalent ionic strength (e.g., NH₄AC), or a combinationof salts, may be substituted. The formula for ionic strength is wellknown to those of skill in the art:

${I = {\frac{1}{2}{\sum\limits_{i = 1}^{n}{c_{i}z_{i}^{2}}}}},$

where c_(i) is the molar concentration of ion i (M, mol/L), z_(i) is thecharge number of that ion, and the sum is taken over all ions in thesolution. For a 1:1 electrolyte such as sodium chloride (NaCl),potassium chloride (KCl), formate (HCO₂ ⁻), or acetate (CH₂CO₂ ⁻) (e.g.,NH₄Ac or NaAc), the ionic strength is equal to the concentration.However, for a sulfate (SO₄ ²⁻), the ionic strength is four timeshigher. Thus, where reference is made to a specific concentration ofNaCl, or a range of concentrations, one of skill in the art cansubstitute another salt, or a mixture of suitable salts, adjusted to theappropriate concentration to provide an ionic strength equivalent tothat provided for NaCl. As used herein this this may be termed a “saltequivalent”, e.g., “NaCl or equivalent”. This will be understood toinclude both a single salt, a mixture of NaCl with other salts, or amixture of salts which do not include NaCl, but which are compatiblewith the apparatus and processes (e.g., affinity and/or anion exchangeresin processes) described herein.

The novel FPLC strategy provided herein utilizes a strong anion exchangeresin complex as described herein. The anion exchange resin binds therAAV9 empty and full capsids are bound by a charge interaction while inbuffer A (the running buffer). In one embodiment, the anion exchangeresin column in equilibrated using Buffer A which contains about 200 nMNaCl to about 700 nM NaCl, or about 400 mM NaCl to about 650 mM NaCl, orsalt equivalent. Suitable buffers may include ions contributed from avariety of sources, such as, e.g., N-methylpiperazine; piperazine;Bis-Tris; Bis-Tris propane; MES, Hepes, BTP or a phosphate bufferN-methyldiethanolamine; 1,3-diaminopropane; ethanolamine; acetic acidand the like. Such buffers are generally used at a neutral pH (e.g.,about 6.5 to about 8, preferably, about 7 to about 7.5, or about 7.5).In one embodiment, a Tris buffer component is selected. In oneembodiment, Buffer A contains about 20 mM Tris-Cl, about 400 nM NaCl orequivalent, pH 7.5.

The rAAV particles and intermediates become dissociated and returns tosolution (suspension) in buffer B (the elution buffer). Buffer B is usedto equilibrate the anion exchange resin. As provided herein, Buffer B ispreferably at a pH of 10.2. While less optimal, the pH may be adjustedas low as about 10.0 or as high as about 10.4. In one embodiment, thebuffer contains about 20 mM Bis-Tris Propane (BTP) and about 10 mM NaClto about 40 nM NaCl (or salt equivalent).

A mixture containing rAAV9 empty and full particles may be suspended inabout 100% Buffer A and applied to the column (vessel). The rAAV9particles and intermediates bind to the resin while other components arecarried out in the buffer. In one embodiment, the total flow rate of thebuffer is kept constant; however, the proportion of Buffer B (the“elution” buffer) is gradually increased from 0% to 100% according to aprogrammed change in concentration (the “gradient”).

In one embodiment, at least one nuclease digestion step is performedprior to loading the mixture onto the anion exchange resin, i.e., duringthe harvest of the rAAV particles and intermediates from the productioncell culture. In a further embodiment, a second nuclease digestion step(e.g., Benzonase) is performed prior to loading the mixture onto theanion exchange resin. Suitably, this may be performed during affinitycapture. For example, an additional wash step may be incorporated intothe affinity method in which the selected nuclease(s) are pre-mixed witha buffer and used in a wash step. Suitably, the buffer is at neutral pHand a relatively low salt concentration, e.g., about 10 to about 100 mM,about 20 mM to about 80 mM, about 30 mM NaCl to about 50 mL, or about 40mM, based on the ionic strength of NaCl or a salt equivalent to any ofthe preceding ranges or amounts. In one embodiment, the flow rate forthis wash step is performed at a slower rate than the other wash stepsto allow for greater exposure of the nuclease to the loaded rAAVparticles and intermediates.

In one embodiment, the salt gradient has an ionic strength equivalent toat least about 10 mM NaCl to about 200 mM NaCl or salt equivalent. Inanother embodiment the salt gradient has an ionic strength equivalent toat least about 40 mM to about 190 mM NaCl, or about 70 nM to about 170nM NaCl. In one embodiment, the AAV9 intermediates are separated fromthe anion exchange resin when the salt gradient reaches an ionicstrength equivalent to about 50 nM NaCl or greater, or about 70 nM NaClor greater.

At different points during this process, as described herein, the boundrAAV9 particles and rAAV9 empty intermediates dissociate and appear inthe effluent. The effluent passes through two detectors which measuresalt concentration (by conductivity) and protein concentration (byabsorption of ultraviolet light at a predetermined wavelength). However,other suitable detection means may be used. As each protein is eluted itappears in the effluent as a “peak” in protein concentration and can becollected for further use.

As described herein, the fractions under the 260nm elution peakcontaining the rAAV9 viral particles (“full”) are collected andprocessed for further use. In one embodiment, the resulting rAAV9preparation or stock contains a ratio of particles to vector genomesof 1. Optionally, the rAAV9 viral particles are placed in a suspensionhaving a pH closer to a neutral pH which will be used for long-termstorage and/or delivery to patients. Such a pH may be in the range ofabout 6.5 to about 8, or about 7 to about 7.5.

In one embodiment, particles elute in a pH of about 10.2 and the rAAVparticles are at least about 50% to about 90% purified from AAV9intermediates, or a pH of 10.2 and about 90% to about 99% purified fromAAV9 intermediates. A stock or preparation of rAAV9 particles (packagedgenomes) is “substantially free” of AAV empty capsids (and otherintermediates) when the rAAV9 particles in the stock are at least about75% to about 100%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, or at least 99% of the rAAV9 in the stockand “empty capsids” are less than about 1%, less than about 5%, lessthan about 10%, less than about 15% of the rAAV9 in the stock orpreparation.

In a further embodiment, the average yield of rAAV particles from loadedmaterial is at least about 70%. This may be calculated by determiningtiter (genome copies) in the mixture loaded onto the column and theamount presence in the final elutions. Further, these may be determinedbased on q-PCR analysis and/or SDS-PAGE techniques such as thosedescribed herein (see figure legends) or those which have been describedin the art.

For example, to calculate empty and full particle content, VP3 bandvolumes for a selected sample (e.g., in examples herein an iodixanolgradient-purified preparation where # of GC=# of particles) are plottedagainst GC particles loaded. The resulting linear equation (y=mx+c) isused to calculate the number of particles in the band volumes of thetest article peaks. The number of particles (pt) per 20 μL loaded isthen multiplied by 50 to give particles (pt) /mL. Pt/mL divided by GC/mLgives the ratio of particles to genome copies (pt/GC). Pt/mL-GC/mL givesempty pt/mL. Empty pt/mL divided by pt/mL and x 100 gives the percentageof empty particles.

Generally, methods for assaying for empty capsids and AAV vectorparticles with packaged genomes have been known in the art. See, e.g.,Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec.Ther. (2003) 7:122-128. To test for denatured capsid, the methodsinclude subjecting the treated AAV stock to SDS-polyacrylamide gelelectrophoresis, consisting of any gel capable of separating the threecapsid proteins, for example, a gradient gel containing 3-8%Tris-acetate in the buffer, then running the gel until sample materialis separated, and blotting the gel onto nylon or nitrocellulosemembranes, preferably nylon. Anti-AAV capsid antibodies are then used asthe primary antibodies that bind to denatured capsid proteins,preferably an anti-AAV capsid monoclonal antibody, most preferably theB1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000),9281-9293). A secondary antibody is then used, one that binds to theprimary antibody and contains a means for detecting binding with theprimary antibody, more preferably an anti-IgG antibody containing adetection molecule covalently bound to it, most preferably a sheepanti-mouse IgG antibody covalently linked to horseradish peroxidase. Amethod for detecting binding is used to semi-quantitatively determinebinding between the primary and secondary antibodies, preferably adetection method capable of detecting radioactive isotope emissions,electromagnetic radiation, or colorimetric changes, most preferably achemiluminescence detection kit. For example, for SDS-PAGE, samples fromcolumn fractions can be taken and heated in SDS-PAGE loading buffercontaining reducing agent (e.g., DTT), and capsid proteins were resolvedon pre-cast gradient polyacylamide gels (e.g., Novex). Silver stainingmay be performed using SilverXpress (Invitrogen, Calif.) according tothe manufacturer's instructions. In one embodiment, the concentration ofAAV vector genomes (vg) in column fractions can be measured byquantitative real time PCR (Q-PCR). Samples are diluted and digestedwith DNase I (or another suitable nuclease) to remove exogenous DNA.After inactivation of the nuclease, the samples are further diluted andamplified using primers and a TagMan™ fluorogenic probe specific for theDNA sequence between the primers. The number of cycles required to reacha defined level of fluorescence (threshold cycle, Ct) is measured foreach sample on an Applied Biosystems Prism 7700 Sequence DetectionSystem. Plasmid DNA containing identical sequences to that contained inthe AAV vector is employed to generate a standard curve in the Q-PCRreaction. The cycle threshold (Ct) values obtained from the samples areused to determine vector genome titer by normalizing it to the Ct valueof the plasmid standard curve, End-point assays based on the digital PCRcan also be used.

In one aspect, an optimized q-PCR method is provided herein whichutilizes a broad spectrum serine protease, e.g., proteinase K (such asis commercially available from Qiagen). More particularly, the optimizedqPCR genome titer assay is similar to a standard assay, except thatafter the DNase I digestion, samples are diluted with proteinase Kbuffer and treated with proteinase K followed by heat inactivation.Suitably samples are diluted with proteinase K buffer in an amount equalto the sample size. The proteinase K buffer may be concentrated to 2fold or higher. Typically, proteinase K treatment is about 0.2 mg/mL,but may be varied from 0.1 mg/mL to about 1 mg/mL. The treatment step isgenerally conducted at about 55° C. for about 15 minutes, but may beperformed at a lower temperature (e.g., about 37° C. to about 50° C.)over a longer time period (e.g., about 20 minutes to about 30 minutes),or a higher temperature (e.g., up to about 60° C.) for a shorter timeperiod (e.g., about 5 to 10 minutes). Similarly, heat inactivation isgenerally at about 9.5 ° C. for about 15 minutes, but the temperaturemay be lowered (e.g., about 70 to about 90° C.) and the time extended(e.g., about 20 minutes to about 30 minutes). Samples are then diluted(e.g., 1000 fold) and subjected to TaqMan analysis as described in thestandard assay.

Additionally, or alternatively, droplet digital PCR (ddPCR) may be used.For example, methods for determining single-stranded andself-complementary AAV vector genome titers by ddPCR have beendescribed. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum GeneTher Methods. 2014 April;25(2): 15-25. doi: 10.1089/hgtb.2013.131. Epub2014 Feb. 14.

In one embodiment, the mixture which is applied to the anion exchangeresin has been purified from contamination with materials present fromthe production system. Suitably, the mixture comprising the recombinantAAV9 viral particles and AAV9 intermediates contains less than about 10%contamination from non-AAV viral and cellular proteinaceous and nucleicacid materials, or less than about 5% contaminants, or less than 1%contaminating viral and cellular proteinaceous and nucleic acidmaterials. Thus, the mixture loaded onto the anion exchange resin isabout 95% to about 99% free of contaminants.

As used herein, the term “contaminants” refer to host cell, viral, andother proteinaceous materials which are present in the productionculture or are by-products thereof. This term does not include rAAVparticles or rAAV intermediates having formed AAV capsids.

In one embodiment, the invention utilizes a two-step chromatographymethod in which affinity capture is utilized to separate a mixture ofrecombinant AAV9 viral particles and AAV 9 capsid intermediates fromproduction system contaminants. Advantageously, this processing has beenfound to allow approximately 3 times to 5 times the amount of startingmaterial (based on the concentration of rAAV genome copies) to beprocessed using approximately 5 to 10 less resin, as compared to certainprior art approaches (e.g., one prior art approach utilized affinitycapture after anion exchange and another utilized multiple, sequential,ion exchange resin columns).

This affinity capture is suitably performed using an antibody-captureaffinity resin. In one embodiment, the solid support is a cross-linkedpoly(styrene-divinylbenzene) having an average particle size of about 50μm and having an AAV-specific antibody. An example of one suchcommercially available affinity resin is POROS™ high performanceaffinity resin commercially available from Thermo Fischer Scientific.The resin contains ligands created by a proprietary technology based oncamelid-derived single-domain antibody fragments coupled to the resinvia carbonyldiimidazole (CDI). The ligand is a 13-kDa single-domainfragment that comprises the 3 CDRs that form the antigen binding domainand is efficiently produced by the yeast Saccharomyces cerevisiae in aproduction process free of animal components. Other suitable affinityresins may be selected or designed which contain an AAV-specificantibody, AAV9 specific antibody, or other immunoglobulin constructwhich is an AAV-specific ligand. Such solid supports may be any suitablepolymeric matrix material, e.g., agarose, sepharose, sephadex, amongstothers.

Suitable loading amounts may be in the range of about 2 to about 5×10¹²GC/mL resin, or less. Equivalent amounts may be calculated for othersized columns or other vessels. At this point prior to anion exchangeresin separation as described herein, the term “genome copy” refers tothe full particles in a mixture of both rAAV9 full particles and rAAV9empties/intermediates.

In one embodiment, the mixture is buffer exchanged with the columnequilibration/loading buffer. The method described herein utilizes arelatively high salt concentration for loading the column. In oneembodiment, the mixture containing the AAV9 viral particles and AAV9intermediates is loaded onto the affinity resin in a buffer having ahigh salt concentrations, e.g., about 400 nM NaCl to about 650 mM NaClor another salt(s) having an equivalent ionic strength. The wash stepfor the affinity resin is thereafter performed at an even higher saltconcentration, e.g., in the range of about 750 mM to about 1 M NaCl orequivalent. In one embodiment, the AAV9 mixture is maintained at a saltconcentration of about 400 mM NaCl to about 650 mM NaCl, or equivalentprior to being applied to the anion exchange resin column. In a furtherembodiment, the rAAV9 mixture is maintained at this salt concentrationfollowing concentration and prior to loading onto the affinity resin.One example of a suitable buffer is Buffer A, containing about 200 nM toabout 600 nM NaCl, or about 400 nM NaCl, or the ionically equivalent ofanother salt, about 10 mM to about 40 mM Tris-Cl or another buffer, at aneutral pH. The flow rate at loading may be a manufacturer's recommendedvalue, e.g., about 149 cm/hr. A wash step using Buffer C is applied (1 MNaCl or an equivalent salt, 20 mM sodium citrate, neutral pH), followedby a wash with Buffer A, and use of Buffer B for elution. In oneembodiment, Buffer B is about 200 nM to about 600 nM NaCl, or about 400nM NaCl, or the ionically equivalent of another salt, about 10 mM toabout 40 mM Tris-Cl, or about 20 nM Tris-Cl or another buffer. In oneembodiment, this step is performed at the range recommended by themanufacturer, e.g., a low pH such as, e.g., about 2.5. In oneembodiment, about 2 to about 8, or about 5 column volumes of buffer areused for these steps.

In one embodiment, at least one nuclease digestion step is performedprior to loading the mixture onto the anion exchange resin, i.e. ,during the harvest of the rAAV particles and intermediates from theproduction cell culture. In a further embodiment, a second nucleasedigestion step is performed during affinity capture. For example, anadditional wash step may be incorporated into the affinity method inwhich the selected nuclease(s) are pre-mixed with a buffer and used in awash step. Suitably, the buffer is at neutral pH and a relatively lowsalt concentration, e.g., about 20 to about 60 mM, about 30 mM NaCl toabout 50 mL, or about 40 mM, based on the ionic strength of NaCl or asalt equivalent to any of the preceding ranges or amounts. In oneembodiment, the flow rate for this wash step is performed at a slowerrate than the other wash steps to allow for greater exposure of thenuclease to the loaded rAAV particles and intermediates.

A single nuclease, or a mixture of nucleases, may be used in this step.Such nucleases may target single stranded DNA, double-stranded DNA, orRNA. While the working examples illustrate use of a deoxyribonuclease(DNase) (e.g., Benzonase or Turbonuclease), other suitable nucleases areknown, many of which are commercially available. Thus, a suitablenuclease or a combination of nucleases, may be selected. Further, thenuclease(s) selected for this step may be the same or different from thenuclease(s) used during the processing preceding the affinity step andwhich more immediately follows harvest from the cell culture.

In one embodiment, the load for the first affinity chromatography stepis obtained following harvest and subsequent processing of cell lysatesand/or supernatant of a production cell culture. This processing mayinvolve at least one of the following processes, including, optionallysis, optional collection from supernatant (media), filtrations,clarification, concentration, and buffer exchange.

Numerous methods are known in the art for production of rAAV vectors,including transfection, stable cell line production, and infectioushybrid virus production systems which include Adenovirus-AAV hybrids,herpesvirus-AAV hybrids and baculovirus-AAV hybrids. See, e.g., G Ye, etal, Hu Gene Ther Clin Dev, 25: 212-217 (December 2014); RM Kotin, Hu MolGenet, 2011, Vol. 20, Rev Issue 1, R2-R6; M. Mietzsch, et al, Hum GeneTherapy, 25: 212-222 (March 2014); T Virag et al, Hu Gene Therapy, 20:807-817 (August 2009); N. Clement et al, Hum Gene Therapy, 20: 796-806(August 2009); DL Thomas et al, Hum Gene Ther, 20: 861-870 (August2009). rAAV production cultures for the production of rAAV virusparticles all require; 1) suitable host cells, including, for example,human-derived cell lines such as HeLa, A549, or 293 cells, orinsect-derived cell lines such as SF-9, in the case of baculovirusproduction systems; 2) suitable helper virus function, provided by wildtype or mutant adenovirus (such as temperature sensitive adenovirus),herpes virus, baculovirus, or a nucleic acid construct providing helperfunctions in trans or in cis; 3) functional AAV rep genes, functionalcap genes and gene products; 4) a transgene (such as a therapeutictransgene) flanked by AAV ITR sequences; and 5) suitable media and mediacomponents to support rAAV production.

A variety of suitable cells and cell lines have been described for usein production of AAV. The cell itself may be selected from anybiological organism, including prokaryotic (e.g., bacterial) cells, andeukaryotic cells, including, insect cells, yeast cells and mammaliancells. Particularly desirable host cells are selected from among anymammalian species, including, without limitation, cells such as A549,WEHI, 3T3, 10T1/2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO,WI38, HeLa, a HEK 293 cell (which express functional adenoviral E1),Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyteand myoblast cells derived from mammals including human, monkey, mouse,rat, rabbit, and hamster. In certain embodiments, the cells aresuspension-adapted cells. The selection of the mammalian speciesproviding the cells is not a limitation of this invention; nor is thetype of mammalian cell, i.e., fibroblast, hepatocyte, tumor cell, etc.

AAV sequences may be obtained from a variety of sources. For example, asuitable AAV sequence may be obtained as described in WO 2005/033321 orfrom known sources, e.g., the American Type Culture Collection, or avariety of academic vector core facilities. Alternatively, suitablesequences are synthetically generated using known techniques withreference to published sequences. Examples of suitable AAV sequences areprovided herein.

In addition to the expression cassette, the cell contains the sequenceswhich drive expression of an AAV capsid in the cell (cap sequences) andrep sequences of the same source as the source of the AAV ITRs found inthe expression cassette, or a cross-complementing source. The AAV capand rep sequences may be independently selected from different AAVparental sequences and be introduced into the host cell in a suitablemanner known to one in the art. While the full-length rep gene may beutilized, it has been found that smaller fragments thereof, i.e., therep78/68 and the rep52/40 are sufficient to permit replication andpackaging of the AAV.

In one embodiment, the host cell contains at least the minimumadenovirus DNA sequences necessary to express an E1a gene product, anE1b gene product, an E2a gene product, and/or an E4 ORF6 gene product.In embodiments in which the host cell carries only E1, the E2a geneproduct and/or E4 ORF6 gene product may be introduced via helper plasmidor by adenovirus co-infection. In another embodiment, the E2a geneproduct and/or E4 ORF6 may be substituted by herpesvirus helperfunctions. The host cell may contain other adenoviral genes such as VAIRNA, but these genes are not required. In one embodiment, the cell useddoes not carry any adenovirus gene other than E1, E2a and/or E4 ORF6;does not contain any other virus gene which could result in homologousrecombination of a contaminating virus during the production of rAAV;and it is capable of infection or transfection by DNA and expresses thetransfected gene (s).

One cell useful in the present invention is a host cell stablytransformed with the sequences encoding rep and cap, and which istransfected with the adenovirus E1, E2a, and E4ORF6 DNA and a constructcarrying the expression cassette as described above. Stable rep and/orcap expressing cell lines, such as B-50 (International PatentApplication Publication No. WO 99/15685), or those described in U.S.Pat. No. 5,658,785, may also be similarly employed. Another desirablehost cell contains the minimum adenoviral DNA which is sufficient toexpress E4 ORF6. Yet other cell lines can be constructed using the novelmodified cap sequences of the invention.

The preparation of a host cell according to this invention involvestechniques such as assembly of selected DNA sequences. This assembly maybe accomplished utilizing conventional techniques. Such techniquesinclude cDNA and genomic cloning, which are well known and are describedin Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, N.Y., including polymerase chainreaction, synthetic methods, and any other suitable methods whichprovide the desired nucleotide sequence.

The required components for AAV production (e.g., adenovirus E1a, E1b,E2a, and/or E4ORF6 gene products, rep or a fragment(s) thereof, cap, theexpression cassette, as well as any other desired helper functions), maybe delivered to the packaging host cell separately, or in combination,in the form of any genetic element which transfer the sequences carriedthereon.

Alternatively, one or more of the components required to be cultured inthe host cell to package an expression cassette in an AAV capsid may beprovided to the host cell in trans using a suitable genetic element.

Suitable media known in the art may be used for the production of rAAVvectors. These media include, without limitation, media produced byHyclone Laboratories and JRH including Modified Eagle Medium (MEM),Dulbecco's Modified Eagle Medium (DMEM), custom formulations such asthose described in U.S. Pat. No. 6,566,118, and Sf-900 II SFM media asdescribed in U.S. Pat. No. 6,723,551, each of which is incorporatedherein by reference in its entirety, particularly with respect to custommedia formulations for use in production of recombinant AAV vectors.

rAAV production culture media may be supplemented with serum orserum-derived recombinant proteins at a level of 0.5%-20% (v/v or w/v).Alternatively, as is known in the art, rAAV vectors may be produced inserum-free conditions which may also be referred to as media with noanimal-derived products. One of ordinary skill in the art may appreciatethat commercial or custom media designed to support production of rAAVvectors may also be supplemented with one or more cell culturecomponents know in the art, including without limitation glucose,vitamins, amino acids, and or growth factors, in order to increase thetiter of rAAV in production cultures.

rAAV production cultures can be grown under a variety of conditions(over a wide temperature range, for varying lengths of time, and thelike) suitable to the particular host cell being utilized. As is knownin the art, rAAV production cultures include attachment-dependentcultures which can be cultured in suitable attachment-dependent vesselssuch as, for example, roller bottles, hollow fiber filters,microcarriers, and packed-bed or fluidized-bed bioreactors. rAAV vectorproduction cultures may also include suspension-adapted host cells suchas HeLa, 293, and SF-9 cells which can be cultured in a variety of waysincluding, for example, spinner flasks, stirred tank bioreactors, anddisposable systems such as the Wave bag system.

rAAV vector particles of the invention may be harvested from rAAVproduction cultures by lysis of the host cells of the production cultureor by harvest of the spent media from the production culture, providedthe cells are cultured under conditions known in the art to causerelease of rAAV particles into the media from intact cells, as describedmore fully in U.S. Pat. No. 6,566,118). Suitable methods of lysing cellsare also known in the art and include for example multiple freeze/thawcycles, sonication, microfluidization, and treatment with chemicals,such as detergents and/or proteases.

At harvest, rAAV production cultures of the present invention maycontain one or more of the following: (1) host cell proteins; (2) hostcell DNA; (3) plasmid DNA; (4) helper virus; (5) helper virus proteins;(6) helper virus DNA; and (7) media components including, for example,serum proteins, amino acids, transferrins and other low molecular weightproteins.

In some embodiments, the rAAV production culture harvest is clarified toremove host cell debris. In some embodiments, the production cultureharvest is clarified by filtration through a series of depth filtersincluding, for example, a grade DOHC Millipore Millistak+HC Pod Filter,a grade A1HC Millipore Millistak+HC Pod Filter, and a 0.2 μm FilterOpticap XL10 Millipore Express SHC Hydrophilic Membrane filter.Clarification can also be achieved by a variety of other standardtechniques known in the art, such as, centrifugation or filtrationthrough any cellulose acetate filter of 0.2 μm or greater pore sizeknown in the art. Still other suitable depth filters, e.g., in the rangeof about 0.045 μm to about 0.2 μm or other filtration techniques may beused.

Suitably, the rAAV production culture harvest is treated with anuclease, or a combination of nucleases, to digest any contaminatinghigh molecular weight nucleic acid present in the production culture.The examples herein illustrate a DNAse, e.g., Benzonase® digestionperformed under standard conditions known in the art. For example, afinal concentration of 1 unit/mL to 2.5 units/mL of Benzonase® is usedat a temperature ranging from ambient temperature to 37° C. for a periodof 30 minutes to several hours, or about 2 hours. In another example, aturbonuclease is used. However, one of skill in the art may utilizeother another suitable nuclease, or a mixture of nucleases. Examples ofother suitable nuclease is described earlier in this specification.

The mixture containing full rAAV particles and rAAV intermediates(including empty capsids) may be isolated or purified using one or moreof the following purification steps: tangential flow filtration (TFF)for concentrating the rAAV particles, heat inactivation of helper virus,rAAV capture by hydrophobic interaction chromatography, buffer exchangeby size exclusion chromatography (SEC), and/or nanofiltration. Thesesteps may be used alone, in various combinations, or in differentorders. In some embodiments, the method comprises all the steps in theorder as described below.

In some embodiments, the Benzonase®-treated mixture is concentrated viatangential flow filtration (“TFF”). Large scale concentration of virusesusing TFF ultrafiltration has been described by R. Paul et al., HUMANGENE THERAPY, 4:609-615 (1993). TFF concentration of the feedstreamenables a technically manageable volume of feedstream to be subjected tothe chromatography steps of the present invention and allows for morereasonable sizing of columns without the need for lengthy recirculationtimes. In some embodiments, the rAAV feedstream is concentrated betweenat least two-fold and at least ten-fold. In some embodiments, thefeedstream is concentrated between at least ten-fold and at leasttwenty-fold. In some embodiments, the feedstream is concentrated betweenat least twenty-fold and at least fifty-fold. One of ordinary skill inthe art will also recognize that TFF can also be used at any step in thepurification process where it is desirable to exchange buffers beforeperforming the next step in the purification process.

As used herein, the singular form of the articles “a,” “an,” and “the”includes plural references unless indicated otherwise. For example, thephrase “a virus particle” includes one or more virus particles.

As used herein, the terms “comprise”, “comprising”, “contain”,“containing”, and their variants are open claim language, i.e., arepermissive of additional elements. In contrast, the terms “consists”,“consisting”, and its variants are closed claim language, i.e.,exclusive additional elements.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” In the context of pH values, “about” refers to a variability of±0.2 from the given value. For example, “about 10.2” encompasses to 10.0to 10.4. As to other values, unless otherwise specified “about” refersto a variability of ±10% from a given value. In certain embodiments, thevariability may be 1%, 5%, 10%, or values therebetween.

While the purification methods described herein are designedparticularly for separating full rAAV9 particles from empty rAAV9intermediates, one of skill in the art may apply these techniques toother rAAV which are closely related to AAV9, including, e.g., hu31 andhu31, which are described in U.S. Pat. No. 7,906,111, as well as, e.g.,and in particular; AAV having about 99% identity thereto over thefull-length VP1, VP2 or VP3 protein of the AAV9 capsid as definedherein, and/or 100% identity with the AAV9 capsid over the antibodybinding region for the affinity resin. Suitably, AAV the integrity ofthe ligand-binding site for the affinity capture purification ismaintained and the change in sequences does not substantially alter thepH range for the capsid for the ion exchange resin purification. SuchAAV9 variants may include those described, e.g., in U.S. Pat. No.9,102,949, U.S. Pat. No. 8,927,514, US2015/349911; and WO 2016/049230A1.

In still another aspect, the invention provides a scalable method forseparating full AAV9 viral particles from AAV9 intermediates by using ananti-AAV antibody based affinity capture resin followed by an anionexchange resin. In one embodiment, the mixture containing the AAV9 viralparticles and AAV9 intermediates is loaded onto the affinity resin in abuffer having a high salt concentrations, e.g., about 400 nM

NaCl to about 650 mM NaCl or another salt(s) having an equivalent ionicstrength. The wash step for the affinity resin is thereafter performedat an even higher salt concentration, e.g., in the range of about 750 mMto about 1 M NaCl or equivalent. In one embodiment, the AAV9 mixture ismaintained at a salt concentration of about 400 mM NaCl to about 650 mMNaCl, or equivalent prior to being applied to the anion exchange resincolumn. In one embodiment, the affinity capture includes a nucleasedigestion step. In a further embodiment, the rAAV9 mixture is maintainedat this salt concentration following concentration and prior to loadingonto the affinity resin.

In a further embodiment, the affinity purified mixture containing theviral particles having packaged genomic sequences are separated fromgenome-deficient AAV9 capsid intermediates by subjecting the mixture tofast performance liquid chromatography at a pH of about 10.2. Moreparticularly, the AAV9 viral particles and AAV9 intermediates are boundto an anion exchange resin equilibrated at a pH of about 10.2, andsubjected to a salt gradient while monitoring eluate for ultravioletabsorbance at about 260 and about 280, wherein the AAV9 full capsids arecollected from a fraction which is eluted when the ratio of A260/A280reaches an inflection point.

In one aspect, a method for separating AAV9 viral particles from AAV9capsid intermediates is provided which involves:

-   -   (a) mixing a suspension comprising recombinant AAV9 viral        particles and AAV 9 capsid intermediates and a Buffer A        comprising 20mM to 50 mM Bis-Tris propane (BTP) and a pH of        about 10.2;    -   (b) loading the suspension of (a) onto a strong anion exchange        resin column;    -   (c) washing the loaded anion exchange resin with Buffer 1% B        which comprises a salt having the ionic strength of 10 mM to 40        mM NaCl and BTP with a pH of about 10.2;    -   (d) applying an increasing salt concentration gradient to the        loaded and washed anion exchange resin, wherein the salt        gradient is the equivalent of about about 10 mM to about 400 mM        NaCl, or about 10 mM to about 200 mM, or about 10 mM to about        190 mM; and    -   (e) collecting rAAV9 particles from elute obtained at a salt        concentration equivalent to at least 70 mM NaCl, where the rAAV9        particles are at least about 90% purified from AAV9        intermediates.

In one embodiment, the intermediates are eluted from the anion exchangeresin when the salt concentration is the equivalent of greater thanabout 50 mM NaCl. In still a further embodiment, Buffer A is furtheradmixed with NaCl to a final concentration of 1M in order to form orprepare Buffer B. In yet another embodiment, the salt gradient has anionic strength equivalent to 10 mM to about 190 mM NaCl. In still afurther embodiment, the salt gradient has an ionic strength equivalentto 20 mM to about 190 mM NaCl, or about 20 mM to about 170 mM NaCl. Theelution gradient may be from 1% buffer B to about 19% Buffer B.Optionally, the vessel containing the anion exchange resin is a monolithcolumn; loading, washing, and eluting occur in about 60 column volumes.

In still a further embodiment, a method for separating recombinant AAV9viral particles containing DNA comprising genomic sequences fromgenome-deficient (empty) AAV9 capsid intermediates is provided. Themethod involves:

-   -   (a) forming a loading suspension comprising recombinant AAV9        viral particles and empty AAV 9 capsid intermediates which have        been purified to remove non-AAV materials from an AAV producer        cell culture in which the particles and intermediates were        generated; and a Buffer A comprising 20 mM Bis-Tris propane        (BTP) and a pH of about 10.2;    -   (b) loading the suspension of (a) onto a strong anion exchange        resin, said resin being in a vessel having an inlet for flow of        a suspension and/or solution and an outlet permitting flow of        eluate from the vessel;    -   (c) washing the loaded anion exchange resin with Buffer 1% B        which comprises 10mM NaCl and 20mM BTP with a pH of about 10.2;    -   (d) applying an increasing salt concentration gradient to the        loaded and washed anion exchange resin, wherein the salt        gradient ranges from 10 mM to about 190 mM NaCl, inclusive of        the endpoints, or an equivalent; and    -   (e) collecting the rAAV particles from eluate collected        following a salt concentration of at least about 70 mM NaCl, or        an equivalent salt or ionic strength, said rAAV particles being        purified away from 9 intermediates.

In one embodiment, the pH is 10.2 and the rAAV particles are at leastabout 90% purified from AAV9 intermediates. In a further embodiment, theaverage yield of rAAV particles is at least about 70%.

In a further embodiment, the rAAV9 producer cell culture is selectedfrom a mammalian cell culture, a bacterial cell culture, and an insectcell culture, wherein said producer cells comprise at least (i) nucleicacid sequence encoding an AAV9 capsid operably linked to sequences whichdirect expression of the AAV9 capsid in the producer cells; (ii) anucleic acid sequence comprising AAV inverted terminal repeat sequencesand genomic transgene sequences for packaging into the AAV 9 capsid; and(iii) functional AAV rep sequences operably linked to sequences whichdirect expression thereof in the producer cells. In another embodiment,producer cells further comprise helper virus sequences required forpackaging and replication of the AAV9 into a viral particle.

In still another embodiment, the material harvested from the cellculture is applied to an affinity resin to separate contaminants fromAAV9 viral particles and empty AAV9 capsid intermediates.

In a further embodiment, the affinity resin separation comprises:

-   -   (i) equilibrating the affinity resin with Buffer A1 which        comprises about 200 mM to about 600 mM NaCl, about 20 mM Tris-Cl        and a neutral pH prior to applying the material to the affinity        resin;    -   (ii) washing the loaded resin of (a) with Buffer C1 which        comprises about 800 mM NaCl to about 1200 mM NaCl, 20 mM Tris-Cl        and a neutral pH;    -   (iii) washing the Buffer C1-washed resin of (b) with Buffer A1        to reduce salt concentration;    -   (iv) washing the affinity resin of (c) with Buffer B which        comprises about 200 nM to about 600 nM NaCl, 20 mM Sodium        Citrate, pH about 2.4 to about 3; and    -   (v) collecting the eluate of (iv) which comprises the full AAV9        particles and the empty AAV9 capsid fraction for loading onto        the anion exchange resin.

The following examples are illustrative of methods for producing AAVparticles in the supernatant of cell cultures according to the presentinvention.

EXAMPLES

A two-step chromatography purification scheme is described whichselectively captures and isolates the genome-containing AAV vectorparticles from the clarified, concentrated supernatant of HEK 293 cellsfive days post transfection. The load for the first chromatography stepusing an AAV9-specific affinity resin, may consist of filter-clarified,concentrated supernatant harvested from cell culture vessels are treatedwith a nuclease (e.g., Benzonase at 37° C. for 2 hours) followed by ahypertonic shock (e.g., 5 M NaCl for 2 h). Prior to loading, the bulkharvest is buffer-exchanged with the column equilibration/loading buffer(Buffer A) incubated overnight at 4° C., and then filtered with asuitable depth filter (e.g., 0.2 μm PES depth filter (Sartorius)). Thesample is applied to a column according to the following method:

-   -   Equilibration: Buffer A (400 mM NaCl, 20 mM Tris-Cl, pH 7.5)    -   Wash 1: Buffer D (1.5 mM MgCl₂, 40 mM NaCl, 20 mM Tris-Cl, pH        7.5)        -   Premix with 150 μl(37,500 u) Benzonase Nuclease        -   Reduce the flow rate to 30 cm.hr⁻¹ (5 ml/min)    -   Wash 2: Buffer C (1M NaCl, 20 mM Tris-Cl, pH 7.5)    -   Wash 3: Buffer A    -   Elution: Buffer B (400 mM NaCl, 20 mM Sodium Citrate, pH 2.5)    -   Re-equilibration: Poros-9 Buffer A

A volume of 500 μl of Neutralization Buffer (0.01% Pluronic F-68, 0.2 MBis-Tris propane, pH10.2) is pre-added to the elution fraction tubes andupon completion of the run, the 5-ml fractions under the main 280-nmelution peak (typically three fractions) are pooled and diluted 50× withAEX Buffer A-10.2 (20 mM Bis-Tris Propane pH 10.2) plus Pluronic F-68(0.001% final) in a polypropylene bottle.

Anion exchange chromatography is subsequently performed to separate thefull or DNA-carrying viral particles from the contaminating emptyparticles in the second step. Specifically, the diluted column eluatefrom the capture step is applied to a pre-equilibrated CIMmultus QA-8 mlmonolith column (BIA Separations) and the following method is run:

-   -   Flow rate: 10 ml/min    -   Equilibration: 20 CV AEX Buffer 1% B (20 mM Bis-Tris Propane pH        10.2, 10 mM NaCl)    -   Sample Application: approx. 800 ml for three diluted POROS 8 or        9 fractions    -   Wash 1: 10 CV AEX Buffer 1% B-10.2    -   Elution: 1-19% AEX Buffer B-10.2 (20 mM Bis-Tris Propane pH        10.2, 1 M NaCl)        -   Linear gradient in 60 CV @ 10-20 ml/min        -   Strip: 20 CV 100% AEX Buffer B-10.2    -   Re-equilibration: 10 CV AEX Buffer 1% B-10.2

A volume of 370 μl of AEX Neutralization Buffer (0.027% Pluronic F-68,1M Bis-Tris pH 6.3) may be pre-added to the elution tubes to minimizeexposure of the vector to the high pH after elution. Finally, the 10-mlfractions under the main 260-nm elution peak are ultimately pooled andconcentrated/diafiltrated with a formulation buffer using a hollow fibermembrane.

Example 1 Separation of Full rAAV9 Vector Particles from Intermediates

AAV9 vector particles produced by triple transfection in HEK293 cells(Lock et al. 2010, Hum Gene Ther, 21(1): 1259-1271) were purified bycentrifugation through an iodixanol density gradient and the bandrepresenting full (genome-containing) vector particles was isolated. Thevector was reconstituted into a 20 mM Bis-Tris-propane (BTP) buffer A atpH 10.2 and 3×10¹² vector genome copies (GC) of the material was loadedonto a 0.1 mL CIMac-QA™ column (Bia Separations) at 2 mL/min. The columnwas washed in buffer A with 20mM NaCl, eluted with a shallow (20-180mMNaCl, 60 CV) salt gradient at the same flow-rate and then stripped withhigh salt Buffer B (20mM BTP, 1M NaCl). A chromatogram of the CIMac-QA™run is shown in FIG. 1 a. A single peak (P2) was observed in the elutiongradient and notably the A260/A280 ratio of the peak was greater thanone, as would be expected for a pure particle population containingvector genomes.

In a separate experiment, AAV9 empty (genome-deficient) particles wereproduced by the plasmid transfection method of AAV production in 293cells (Lock et al. 2010, Hum Gene Ther, 21(1): 1259-1271), except thatthe plasmid containing the vector genome was omitted. Empty particleswere purified by centrifugation through a CsCl gradient andreconstituted into a Bis-Tris-propane (BTP) buffer A at pH 10.2. Anominal volume (100 uL) of this material was loaded onto a 0.1 mL CIMac-QA column (Bia Separations) and run under identical conditions to thefull AAV9 vector described above. The chromatogram of the CIMac-QA runis shown in FIG. 1 b. Two small peaks (P1 and P3) were observed in theelution gradient and a third (P4) in the high salt column strip; in allthree cases the A260/280 ratio of the peaks was less than one reflectingthe lack of a vector genome component. The overlay of the A280 traces ofthe full and empty chromatograms (FIGS. 1a and 1b ) is shown in FIG. 1cand suggests separation of the full particle peak (P2) from the emptyparticle peaks P3 and P4. Complete separation of empty particle peak P1from the full particle peak (P2) was not be achieved.

In a further experiment, proof of empty and full particle separation wassought by mixing the empty and full AAV9 vector particle preparations inthe same amounts as were used in the deconstructed runs described above.The profile of the 0.1 mL CIMac-QA run of this mixture under identicalconditions to the previous runs is shown in FIG. 1 d. The full particlepeak (P2) separated from empty particle peaks P3 and P4 with A260/280ratios as expected. The leading edge of the empty particle peak P1 wasdiscernable since the A280 trace was higher than the A260 trace, but asexpected from the deconstructed runs overlay (FIG. 1c ) the bulk of P1was not fully separated from P2.

Example 2 Separation of rAAV9 full Particles from AAV9 Intermediates

Clarified AAV9 vector production culture supernatant (Lock et al. 2010,Hum Gene Ther, 21(1): 1259-1271) was loaded to a PorosAAV9TM affinitycolumn (ThermoFisher) at neutral pH in 400 mM salt and eluted with a lowpH (˜2.5) buffer. The eluate was immediately adjusted to neutral pH andthen diluted 50-fold into a Bis-Tris-propane (BTP) buffer A at pH 10.2.6×10¹² vector genome copies (GC) of the affinity purified vectormaterial was loaded onto a 0.1mL CIMmultus -QA™ column (Bia Separations)at 2mL/min. The column was washed in buffer A with 20 mM NaCl, elutedwith a shallow (20-180 mM NaCl, 60 CV) salt gradient at the sameflow-rate and then stripped with high salt Buffer B (20 mM BTP, 1MNaCl). A chromatogram of this run is shown in FIG. 2. As with thede-constructed runs described in Example 1, four major peaks (P1-P4)with very similar profiles were observed. P2 had an A260/A280 ratiogreater than one and contained the majority of genome copies (GC) asmeasured by polymerase chain reaction (PCR). P3 and P4 contained veryfew genome copies and had A260/A280 ratios indicative of emptyparticles. Once again, the leading edge of P1 is inferred by the factthat the A280 trace is higher than the A260 trace but the A260/A280ratio rapidly inverts as P2 starts to elute. Together with thede-constructed run evidence presented in Example 1, the chromatogramindicates that the bulk of P1 is contained within P2. Overall, the datademonstrate that affinity purified vector contains both full and emptyvector populations and that several empty “intermediates” (e.g.partially packaged, partially assembled capsids) are present. Thepurification method is able to separate full vector particles (P2)completely from two of these intermediates (P3 and P4) but onlypartially from the third (P1).

Example 3 Scalability of the Purification Method

A separate affinity-purified AAV9 vector preparation (performed usingPOROS9™ column under the conditions described herein) containing 5×10¹⁴GC was loaded onto an 8 mL CIMmultus-QA™ column with a flow rateadjusted to 20 mL/min to accommodate the increased scale of thepurification run. Despite the increased amount of vector loaded, asimilar chromatographic profile was obtained with 4 peaks (P1-P4)detected in the elution gradient and the column strip (FIG. 3a ).Analysis of GC content once again showed that the majority of the fullparticles eluted in P2 (Data not shown). An SDS-PAGE-based method toquantify total capsids was developed and the peak fractions furtheranalyzed. In this method, a reference preparation purified by iodixanolgradient purification and known to contain 100% full capsids is seriallydiluted and run on an SDS-PAGE gel alongside a similarly diluted testarticle. The stained gel is scanned and the area under the VP3 capsidprotein peaks is determined. In the case of the full reference standardthe GC number loaded equates to vector particle number and hence astandard curve of particle number versus VP3 band volume can beobtained. The standard curve is used to determine the number ofparticles in the peak fractions and division of this number with the GCnumber loaded gives the pt: GC ratio. The number of empty particles canbe obtained by subtracting GC from total particles and used to calculatethe empty:full (E:F) ratio and the percentage of empty particles in asample. This assay and analysis was performed for the scaled AAV9purification run and the results are presented in FIG. 3b . The resultsconfirm the earlier conclusions (Examples 1 and 2) that P1, P3 and P4are comprised of empty particle populations which likely representintermediates in particle packaging and assembly. P2 on the other handis highly enriched for full particles although there is somecross-contamination with empty particles, most likely from peak P1.Further enrichment of the full particle population and reduction ofempty capsid content might be achieved by sub-fractionation of P2 andavoidance of those fractions at the front portion of the peak. Overall,these results demonstrate the scalability of the purification method andthe robustness of peak separation with scale.

(Sequence Listing Free Text)

The following information is provided for sequences containing free textunder numeric identifier <223>.

SEQ ID NO: (containing free text) Free text under <223> 1 <223>Adeno-associated virus 9 vp1 capsid protein

All publications and references to GenBank and other sequences cited inthis specification are incorporated herein by reference in theirentirety, as are the priority applications U.S. Provisional PatentApplication No. 62/322,071, filed Apr. 13, 2016 and U.S. ProvisionalApplication No. 62/266,357, filed Dec. 11, 2015. While the invention hasbeen described with reference to particularly preferred embodiments, itwill be appreciated that modifications can be made without departingfrom the spirit of the invention.

1. A method for separating AAV9 viral particles having packaged genomicsequences from genome-deficient AAV9 capsid intermediates, said methodcomprising: subjecting a mixture comprising recombinant AAV9 viralparticles and AAV9 capsid intermediates to fast performance liquidchromatography, wherein the AAV9 viral particles and AAV9 intermediatesare bound to an anion exchange resin equilibrated at a pH of about 10.2and subjected to a salt gradient while monitoring eluate for ultravioletabsorbance at about 260 and about 280, wherein the AAV9 full capsids arecollected from a fraction which is eluted when the ratio of A260/A280reaches an inflection point.
 2. The method according to claim 1, whereinthe inflection point is when the ratio of A260/A280 changes from lessthan 1 to greater than
 1. 3. The method according to claim 1, whereinthe salt gradient has an ionic strength equivalent to at least about 20mM to about 190 mM NaCl.
 4. The method according to claim 1, wherein theAAV9 intermediates are eluted from the anion exchange resin when thesalt gradient reaches an ionic strength equivalent to about 50 nM NaClor greater.
 5. The method according to claim 1, wherein the mixturecomprising the recombinant AAV9 viral particles and AAV9 capsidintermediates contains less than about 10% contamination from viral andcellular proteinaceous and nucleic acid materials.
 6. The methodaccording to claim 1, wherein the mixture is at least about 95% purifiedfrom viral and cellular proteinaceous and nucleic acid materials.
 7. Themethod according to claim 1, wherein the method has a sample loadingflow rate less than or equal to the elution flow rate.
 8. The methodaccording to claim 1, wherein the anion exchange resin is a strongresin.
 9. The method according to claim 1, wherein the anion exchangeresin is in a column.
 10. The method according to claim 1, wherein themixture comprising recombinant AAV9 viral particles and AAV 9 capsidintermediates had been purified from production system contaminantsusing affinity capture.
 11. The method according to claim 10, whereinthe affinity capture is performed using an affinity resin.
 12. A methodfor separating AAV9 viral particles from AAV9 capsid intermediates, saidmethod comprising: (a) mixing a suspension comprising recombinant AAV9viral particles and AAV 9 capsid intermediates and a Buffer A having apH of about 10.2; (b) loading the suspension of (a) onto a strong anionexchange resin column; (c) washing the loaded anion exchange resin withBuffer 1% B which comprises a salt having and a pH of about 10.2; (d)applying an increasing salt concentration gradient to the loaded andwashed anion exchange resin, wherein the salt gradient is sufficient toelute the rAAV9 particles; and (e) collecting rAAV9 particles which areat least about 90% purified from AAV9 intermediates.
 13. The methodaccording to claim 12, wherein the recombinant AAV9 viral particles andAAV 9 capsid of step (a) have been affinity purified at a high saltconcentration.
 14. The method according to claim 12 or 13, wherein theanion exchange resin is a quaternary amine ion exchange resin.
 15. Themethod according to claim 14, wherein the anion exchange resin columncomprises trimethylamine and a support matrix comprising poly(glycidylmethacrylate-co-ethylene dimethacrylate).
 16. The method according toclaim 12, wherein Buffer A is further admixed with NaCl to a finalconcentration of 1M in order to form or prepare Buffer B.
 17. The methodaccording to claim 12, wherein the salt gradient is from about 10 mM toabout 190 mM NaCl or a salt equivalent.
 18. The method according toclaim 12, wherein the elution gradient is from 1% buffer B to about 19%Buffer B.
 19. The method according to claim 12, wherein when the vesselanion exchange resin column is a monolith column and where columnloading, washing and elution occur in about 60 column volumes.
 20. Themethod according to claim 12, wherein the elution flow rate is fromabout 10 mL/min to about 40 mL/min.
 21. The method according to claim20, wherein the elution flow rate is about 20 mL/min.
 22. A method forseparating recombinant AAV9 viral particles containing DNA comprisingpharmacologically active genomic sequences from genome-deficient(empty)AAV9 capsid intermediates, said method comprising: (a) forming a loadingsuspension comprising: recombinant AAV9 viral particles and empty AAV9capsid intermediates which have been purified to remove non-AAVmaterials from an AAV producer cell culture in which the particles andintermediates were generated; and a Buffer A comprising 20 mM Bis-Trispropane (BTP) and a pH of about 10.2; (b) loading the suspension of (a)onto a strong anion exchange resin, said resin being in a vessel havingan inlet for flow of a suspension and/or solution and an outletpermitting flow of eluate from the vessel; (c) washing the loaded anionexchange resin with Buffer 1% B which comprises 10mM NaCl and 20 mM BTPwith a pH of about 10.2; (d) applying an increasing salt concentrationgradient to the loaded and washed anion exchange resin, wherein the saltgradient ranges from 10 mM to about 190 mM NaCl, inclusive of theendpoints, or an equivalent; and (e) collecting the rAAV particles fromeluate at the inflection point of A260/A280, said rAAV particles beingpurified away from AAV9 intermediates.
 23. The method according to claim22, wherein the pH is 10.2 and the rAAV particles are at least about 90%purified from AAV9 intermediates.
 24. The method according to claim 22,wherein the average yield of rAAV particles is at least about 40% toabout 70% as measured by GC titer.
 25. The method according to claim 22,wherein the producer cell culture is selected from a mammalian cellculture, a bacterial cell culture, and an insect cell culture, whereinsaid producer cells comprise at least (i) nucleic acid sequence encodingan AAV9 capsid operably linked to sequences which direct expression ofthe AAV9 capsid in the producer cells; (ii) a nucleic acid sequencecomprising AAV inverted terminal repeat sequences and genomic transgenesequences for packaging into the AAV 9 capsid; and (iii) functional AAVrep sequences operably linked to sequences which direct expressionthereof in the producer cells.
 26. The method according to claim 22,wherein material harvested from the cell culture is applied to anaffinity resin to separate contaminants from AAV9 viral particles andempty AAV9 capsid intermediates.
 27. The method according to claim 22,wherein the affinity resin separation comprises: (i) equilibrating theaffinity resin with Buffer A1 which comprises about 200 mM to about 600mM NaCl and a neutral pH prior to applying the material to the affinityresin; (ii) washing the loaded resin of (a) with Buffer C1 whichcomprises about 800 mM NaCl to about 1200 mM NaCl and a neutral pH;(iii) washing the Buffer C1-washed resin of (b) with Buffer A1 to reducesalt concentration; (iv) washing the affinity resin of (c) with Buffer Bwhich comprises about 200 nM to about 600 nM NaCl, 20 mM Sodium Citrate,pH about 2.4 to about 3; and (v) collecting the eluate of (iv) whichcomprises the full AAV9 particles and the empty AAV9 capsid fraction forloading onto the anion exchange resin.
 28. The method according to claim27, wherein the neutral pH is about 7.5.
 29. The method according toclaim 27, wherein in (iv), the pH is about 2.5.
 30. The method accordingto claim 27, wherein the equilibrating (i) Buffer A1 and/or Buffer B,independently have about 400 nM NaCl.